Systems and methods for improved focus tracking using a light source configuration

ABSTRACT

Provided herein is an imaging system including a light source; a first focusing lens positioned to focus a beam from the light source to a beam waist at a predetermined location along an optical path of the beam in the imaging system; a beam splitter positioned relative to the first focusing lens to receive the beam from the first focusing lens and to create first and second beams; a second focusing lens positioned to receive the first and second beams output by the beam splitter, to focus the received first and second beams to a spot sized dimensioned to fall within a sample to be image, and further positioned to receive first and second beams reflected from the sample; an image sensor positioned to receive the light beams reflected from the sample; and a roof prism positioned in the optical path between the second focusing lens and the image sensor.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of and claims the benefit of U.S.application Ser. No. 15/913,903, filed Mar. 6, 2018, and U.S.Provisional Application No. 62/468,353, filed Mar. 7, 2017, which ishereby incorporated herein by reference in its entirety. Thisapplication also claims priority to Netherlands Patent Application No.N2018857, filed on May 5, 2017, which is hereby incorporated herein byreference in its entirety.

BACKGROUND

Numerous advances in the field of biology have benefited from improvedimaging systems and techniques, such as, for example, those used inoptical microscopes and scanners. Obtaining accurate focus duringimaging using these imaging systems can be important for successfulimaging operations. Additionally, decreasing the latency associated withfocusing the system on a sample increases the speed at which the systemcan operate.

Many pre-existing scanning systems use a multi-beam focus track systemto determine focus distances for a given sample. The multi-beam systemfocuses two beams onto the sample using objective lens. The focus beamsare reflected from the surface of the sample and reflected beams aredirected toward an image sensor. Reflected beams form spots on the imagesensor and the distance between the spots can be used to determine thefocus distance.

Designers of pre-existing systems are constantly striving to improvefocus accuracy and the speed with which the system can determine thefocus setting. Improving accuracy can be important in that it can allowthe system to achieve better results. Reducing latency can be animportant consideration because it can allow the system to reach a focusdetermination more quickly, thus allowing the system to completescanning operations faster.

SUMMARY

Various examples of the technologies disclosed herein provide systemsand methods for improving accuracy of focus tracking in optical systems.Further examples provide systems and methods for reducing latencyassociated with focus tracking in optical scanners. In some examples,systems and methods are provided for improving or reducing latencyassociated with focus tracking in optical scanners.

Some examples may include an imaging system with a light source, a firstfocusing lens positioned to focus a beam from the light source to a beamwaist at a predetermined location along an optical path of the beam inthe imaging system; a beam splitter positioned relative to the firstfocusing lens to receive the beam from the first focusing lens and tocreate first and second beams; a second focusing lens positioned toreceive the first and second beams output by the beam splitter, to focusthe received first and second beams to a spot sized dimensioned to fallwithin a sample to be image, and further positioned to receive first andsecond beams reflected from the sample; an image sensor positioned toreceive the light beams reflected from the sample; and a roof prismpositioned in the optical path between the second focusing lens and theimage sensor, the roof prism dimensioned to cause the first and secondbeams reflected from the sample to converge on to the image sensor.

Additionally, further examples may include a light source that mayinclude a laser and an optical fiber including first and second ends,the first end of the optical fiber connected to receive output from thelaser, such that the first focusing lens is positioned at apredetermined distance from the second end of the optical fiber.Furthermore, by way of example only, the predetermined distance betweenthe first focusing lens and the second end of the optical fiber may bedetermined using the focal length of the lens to place the beam waist atthe predetermined location along the optical path of the beam.

In further examples, by way of example only, the imaging system mayfurther include a body portion defining a cavity; and an insert slidablymounted within the cavity of the body portion, such that the insertincludes a cylindrical cavity including a first end adjacent the firstfocusing lens and a second end and adjacent the second end of theoptical fiber, further such that the insert is linearly adjustablewithin the cavity to change the distance between the first focusing lensand the second end of the optical fiber. Additionally, the imagingsystem may also include a locking mechanism to fix a position of theinsert within the body portion.

In some instances, by way of example only, the first focusing lens is aPlano convex lens. By way of further example only, the predeterminedlocation along the optical path of the beam for the beam waist may bebetween about 600 mm and 800 mm.

Additionally, the predetermined location along the optical path of thebeam for the beam waist may be chosen such that the spot sizes on theimage sensor of the first and second beams reflected from the sample arewithin 3-10 pixels of each other. In other instances, the spot sizes onthe image sensor of the first and second beams reflected from the sampleare within 4-7 pixels of each other. In other instances, by way offurther example only, the spot sizes on the image sensor of the firstand second beams reflected from the sample are within 5 pixels of eachother.

In some examples, the spot sizes on the image sensor of the first andsecond beams reflected from the sample are no greater than about 330 μmin diameter. Again, by way of example only, spot sizes on the imagesensor of the first and second beams reflected from the sample arewithin a range of about 495 μm in diameter, and in other instances 200μm to 500 μm in diameter

In some examples, the predetermined location along the optical path ofthe beam for the beam waist is chosen such that the spot sizes on theimage sensor of the first and second beams reflected from the sample areno greater than 60 pixels in diameter. In another example, by way ofexample only, the spot sizes on the image sensor of the first and secondbeams reflected from the sample are no greater than 90 pixels indiameter. By way of further example only, the spot sizes on the imagesensor of the first and second beams reflected from the sample arebetween 40 and 90 pixels in diameter.

Additionally, the predetermined location along an optical path of thebeam waist may be within the second focusing lens.

Other features and aspects of the disclosed examples will becomeapparent from the following detailed description, taken in conjunctionwith the accompanying drawings, which illustrate, by way of example, thefeatures in accordance with examples of the invention. The summary isnot intended to limit the scope of the invention, which is definedsolely by the claims attached hereto.

BRIEF DESCRIPTION OF THE DRAWINGS

The technology disclosed herein, in accordance with one or more exampleimplementations, is described in detail with reference to the followingfigures. These figures are provided to facilitate the reader'sunderstanding of the disclosed technology, and are not intended to beexhaustive or to limit the disclosure to the precise forms disclosed.Indeed, the drawings in the figures are provided for purposes ofillustration only, and merely depict example implementations of thedisclosed technology. Furthermore, it should be noted that for clarityand ease of illustration, the elements in the figures have notnecessarily been drawn to scale.

Some of the figures included herein illustrate various examples of thedisclosed technology from different viewing angles. Although theaccompanying descriptive text may refer to such views as “top,” “bottom”or “side” views, such references are merely descriptive and do not implyor require that the disclosed technology be implemented or used in aparticular spatial orientation unless explicitly stated otherwise.

FIG. 1 illustrates a simplified block diagram of one example of an imagescanning system with which systems and methods disclosed herein may beimplemented.

FIGS. 2A and 2B illustrate an example optical system for focus tracking.Particularly, FIG. 2A illustrates an example optical design for focustracking in accordance with one example implementation of the systemsand methods described herein. FIG. 2B is a diagram illustrating analternative view of a portion of the optical system shown in FIG. 2A.

FIG. 3A illustrates an example of a sample container configured to houseone or more samples to be imaged that comprises multiple layers.

FIG. 3B is a diagram illustrating an example of the creation of desiredand unwanted reflections off of the multiple surfaces of a multilayersample container in some environments.

FIG. 3C is a diagram illustrating an example of the effect of theunwanted reflections on the image sensor.

FIG. 3D is a diagram illustrating a reduction in noise at the imagesensor as a result of the placement of blocking structures in accordancewith example applications of the technology disclosed herein.

FIG. 4A illustrates a portion of an image sensor comprising a pluralityof pixels (not shown for clarity of illustration) onto which the focustracking beams are directed in accordance with one exampleimplementation of the systems and methods described herein.

FIG. 4B is a diagram illustrating intensities of left and right focusbeams reflecting onto the image sensor from the S2 and S3 surfaces atdifferent focus settings with the collimating lens adjusted to positiona beam waist along the optical path of the focus tracking beams.

FIG. 4C is a diagram illustrating intensities of the left and rightfocus beams reflecting onto the image sensor from the S2 and S3 surfacesat different focus settings with the collimating lens adjusted toposition at the beam waist more optimally along the optical path of thefocus tracking beams.

FIG. 5A is a diagram illustrating an example of a lens implemented tocause focus tracking beams to converge on a sample plane and be focusedonto an image sensor.

FIG. 5B is a diagram illustrating an example of a roof prism implementedto cause to focus tracking beams to converge onto an image sensor.

FIG. 6 is a diagram illustrating an example configuration including alens positioned to place a beam waist of the focus tracking beam at aselected position.

FIG. 7 is a diagram illustrating an example of a focus tracking systemwith which systems and methods described herein may be implemented.

FIGS. 8 and 9 are diagrams illustrating spatial relationships ofreflected focus tracking beams in one example.

FIG. 10 is a diagram illustrating an example placement of a beam blockerto block reflections of the left and right focus tracking beams from theS4 surface.

FIGS. 11 and 12 are diagrams illustrating the spatial relationship ofreflected focus tracking beams at beam splitter in the exampleconfiguration of FIG. 7, with the beam blocker placed as shown in FIG.10.

FIGS. 13 and 14 illustrate the beams reflected off of the top periscopemirror and the beam splitter in one example.

FIG. 15A is a top-down view illustrating an example of the focustracking beams reflected off surfaces S2 and S4 returned from theobjective lens and directed toward splitter.

FIG. 15B is a close up view of FIG. 15A illustrating how the S4reflected beams can be blocked at the rear face of the splitter by ablocking member.

FIG. 15C is a diagram illustrating a top-down view of an example of ablocking member positioned at the rear face of a splitter.

FIG. 15D is a diagram illustrating a representation of a 4 mm wideblocking structure in the beam path of the reflected focus trackingbeams at the splitter.

FIGS. 16A and 16B are diagrams illustrating an example of a beam blockerthat can be used to block the S4 reflections at the filter/splitter inaccordance with the example implementations described with reference toFIGS. 8-10.

FIG. 17A presents a cutaway view of a beam blocker installed at the beamsplitter in one example.

FIG. 17B presents a rear view of a beam blocker installed at the beamsplitter.

FIG. 18A illustrates an example of an aperture that can be used to blockthe beams reflected off the S1 surface.

FIG. 18B illustrates an example placement of the aperture in front ofthe beam splitter normal to the beam axis.

FIG. 19 shows spots from the beams at a top periscope mirror forfocusing at the top of the sample.

FIG. 20 shows spots from the beams at a top periscope mirror forfocusing at the bottom of the sample.

FIG. 21 illustrates spots at the camera for the S2, S3 reflected beamsfor imaging at the top of the capture range when focusing on S2.

FIG. 22 illustrates spots at the camera for the S2, S3 reflected beamsfor imaging at the bottom of the capture range when focusing on S2.

FIG. 23 illustrates spots at the camera for the S2, S3 reflected beamsfor imaging at the top of the capture range when focusing on S3.

FIG. 24 illustrates spots at the camera for the S2, S3 reflected beamsfor imaging at the bottom of the capture range when focusing on S3.

FIG. 25A illustrates, in one example, spot fringe variation in a beamspot on the image sensor with a laser diode operating in a lasing mode.

FIG. 25B illustrates, in one example, a spot profile in a beam spot onthe image sensor with a laser diode operating in a low-power mode.

FIG. 26 is a diagram illustrating an example of a laser diode operatedin an ASE mode.

FIG. 27 is a diagram illustrating an example of a laser diode operatedin a lasing mode.

FIG. 28 is a diagram illustrating an example of a laser diode operatedin a hybrid mode.

FIG. 29 illustrates instability in spot size when a laser diode ispowered to operate in a lasing mode.

FIG. 30A illustrates an example of spot movement with a laser diodeoperating in a hybrid mode.

FIG. 30B illustrates an example of a spot movement with a laser diodeoperating in a hybrid mode.

FIG. 31 is a diagram illustrating an example of the spectral width ofvarious laser sources tested to determine the relationship betweenspectral width at 5% and set power.

It should be understood that the disclosed technology can be practicedwith modification and alteration, and that the disclosed technology belimited only by the claims and the equivalents thereof.

DETAILED DESCRIPTION

Various example implementations of the technologies disclosed hereinprovide systems and methods for improving or reducing latency associatedwith focus tracking in optical scanners. Various additional examplesprovide systems and methods for improving the accuracy of focus trackingsystems in optical scanners. Still further examples combine aspects ofboth. For example, in some examples, systems and methods are provided toblock stray light caused by unwanted reflections from a sample containerfrom reaching the image sensor and hindering the detection of the focustracking beams. In some applications, a sample container for thescanning system may include a sample layer sandwiched between two ormore other layers. In such applications, the surfaces presented by themulti-layer sample container may each present a reflected beam back tothe objective lens and into the return path of the scanning system.Unwanted reflections, which in some cases may be much stronger thanreflections from the sample layer, can decrease the signal-to-noiseratio at the image sensor making it difficult to detect the actual focustracking beams amid all the other optical noise. Unwanted reflections orscattered beams may also overlap with and coherently interfere withfocus tracking spots at the image sensor, and cause fringes to appear,thereby degrading accuracy of focus tracking. Examples of the systemsand methods disclosed herein can place apertures, blocking bars, orother blocking members at one or more points along the return signalpath to provide optically opaque structures to block the unwanted beamsreflected off of the other surfaces from reaching the image sensor.

As another example, further configurations can include an opticalstructure, such as a lens or other curved or partially curved opticalelement in the optical path to shape the focus tracking laser beams. Invarious examples, this can be implemented by inserting the opticalelement in the optical path prior to the objective lens to position abeam waist within the system. More particularly, in someimplementations, the optical element is positioned in the optical pathat a determined distance from the output of the optical fiber so as toplace a beam waist of the one or more focus tracking beams at a desiredlocation along the optical path. The position of the beam waist alongthe optical path may be chosen such that the resultant spots from thefocus tracking beams reflected off multiple surfaces of interest of thesample container are the same size or substantially the same size as oneanother at the image sensor plane to improve focus tracking accuracy andlatency. In further implementations, an adjustment mechanism can beprovided to adjust the location of the optical element for optimalplacement of the beam waist along the optical path.

As yet another example, further implementations include configurationand adjustment of the optical source for the focus tracking beams. Moreparticularly, some examples may be configured to adjust and set thepower level at which a laser diode source operates to reduce fringing ofthe focus tracking beam spots on the image sensor and to provide a morestable and consistent spot placement. The power level of the laser canbe set such that the laser diode is operating in a quasi-lasing mode orhybrid mode, combining aspects of both an ASE mode of operation and alasing mode of operation. The power level can be set within a range thatis at the high end below the power at which the laser diode emits whatwould normally be considered as highly coherent light, with a singledominant spectral peak and negligible secondary peaks; and at the lowend above the power at which the laser fully emits temporally incoherentlight, also called amplified spontaneous emission (ASE).

Before describing further examples of the systems and methods disclosedherein, it is useful to describe an example environment with which thesystems and methods can be implemented. One such example environment isthat of an image scanning system, such as that illustrated in FIG. 1.The example imaging scanning system may include a device for obtainingor producing an image of a region. The example outlined in FIG. 1 showsan example imaging configuration of a backlight design implementation.

As can be seen in the example of FIG. 1, subject samples are located onsample container 110, which is positioned on a sample stage 170 under anobjective lens 142. Light source 160 and associated optics direct a beamof light, such as laser light, to a chosen sample location on the samplecontainer 110. The sample fluoresces and the resultant light iscollected by the objective lens 142 and directed to a photo detector 140to detect the florescence. Sample stage 170 is moved relative toobjective lens 142 to position the next sample location on samplecontainer 110 at the focal point of the objective lens 142. Movement ofsample stage 110 relative to objective lens 142 can be achieved bymoving the sample stage itself, the objective lens, the entire opticalstage, or any combination of the foregoing. Further examples may alsoinclude moving the entire imaging system over a stationary sample.

Fluid delivery module or device 100 directs the flow of reagents (e.g.,fluorescent nucleotides, buffers, enzymes, cleavage reagents, etc.) to(and through) sample container 110 and waist valve 120. In particularexamples, the sample container 110 can be implemented as a flowcell thatincludes clusters of nucleic acid sequences at a plurality of samplelocations on the sample container 110. The samples to be sequenced maybe attached to the substrate of the flowcell, along with other optionalcomponents.

The system also comprises temperature station actuator 130 andheater/cooler 135 that can optionally regulate the temperature ofconditions of the fluids within the sample container 110. Camera system140 can be included to monitor and track the sequencing of samplecontainer 110. Camera system 140 can be implemented, for example, as aCCD camera, which can interact with various filters within filterswitching assembly 145, objective lens 142, and focusing laser/focusinglaser assembly 150. Camera system 140 is not limited to a CCD camera andother cameras and image sensor technologies can be used.

Light source 160 (e.g., an excitation laser within an assemblyoptionally comprising multiple lasers) or other light source can beincluded to illuminate fluorescent sequencing reactions within thesamples via illumination through fiber optic interface 161 (which canoptionally comprise one or more re-imaging lenses, a fiber opticmounting, etc). Low watt lamp 165, focusing laser 150, and reversedichroic 185 are also presented in the example shown. In some examplesfocusing laser 150 may be turned off during imaging. In other examples,an alternative focus configuration can include a second focusing camera(not shown), which can be a quadrant detector, a Position SensitiveDetector (PSD), or similar detector to measure the location of thescattered beam reflected from the surface concurrent with datacollection.

Although illustrated as a backlit device, other examples may include alight from a laser or other light source that is directed through theobjective lens 142 onto the samples on sample container 110. Samplecontainer 110 can be ultimately mounted on a sample stage 170 to providemovement and alignment of the sample container 110 relative to theobjective lens 142. The sample stage can have one or more actuators toallow it to move in any of three dimensions. For example, in terms ofthe Cartesian coordinate system, actuators can be provided to allow thestage to move in the X, Y and Z directions relative to the objectivelens. This can allow one or more sample locations on sample container110 to be positioned in optical alignment with objective lens 142.

A focus (z-axis) component 175 is shown in this example as beingincluded to control positioning of the optical components relative tothe sample container 110 in the focus direction (referred to as the zaxis, or z direction). Focus component 175 can include one or moreactuators physically coupled to the optical stage or the sample stage,or both, to move sample container 110 on sample stage 170 relative tothe optical components (e.g., the objective lens 142) to provide properfocusing for the imaging operation. For example, the actuator may bephysically coupled to the respective stage such as, for example, bymechanical, magnetic, fluidic or other attachment or contact directly orindirectly to or with the stage. The one or more actuators can beconfigured to move the stage in the z-direction while maintaining thesample stage in the same plane (e.g., maintaining a level or horizontalattitude, perpendicular to the optical axis). The one or more actuatorscan also be configured to tilt the stage. This can be done, for example,so that sample container 110 can be leveled dynamically to account forany slope in its surfaces.

Focusing of the system generally refers to aligning the focal plane ofthe objective lens with the sample to be imaged at the chosen samplelocation. However, focusing can also refer to adjustments to the systemto obtain a desired characteristic for a representation of the samplesuch as, for example, a desired level of sharpness or contrast for animage of a test sample. Because the usable depth of field of the focalplane of the objective lens is very small (sometimes on the order ofabout 1 μm or less), focus component 175 closely follows the surfacebeing imaged. Because the sample container is not perfectly flat asfixtured in the instrument, focus component 175 may be set up to followthis profile while moving along in the scanning direction (referred toas the y-axis).

The light emanating from a test sample at a sample location being imagedcan be directed to one or more detectors 140. Detectors can include, forexample a CCD camera, An aperture can be included and positioned toallow only light emanating from the focus area to pass to the detector.The aperture can be included to improve image quality by filtering outcomponents of the light that emanate from areas that are outside of thefocus area. Emission filters can be included in filter switchingassembly 145, which can be selected to record a determined emissionwavelength and to cut out any stray laser light.

In various applications, sample container 110 can include one or moresubstrates upon which the samples are provided. For example, in the caseof a system to analyze a large number of different nucleic acidsequences, sample container 110 can include one or more substrates onwhich nucleic acids to be sequenced are bound, attached or associated.In various implementations, the substrate can include any inertsubstrate or matrix to which nucleic acids can be attached, such as forexample glass surfaces, plastic surfaces, latex, dextran, polystyrenesurfaces, polypropylene surfaces, polyacrylamide gels, gold surfaces,and silicon wafers. In some applications, the substrate is within achannel or other area at a plurality of locations formed in a matrix orarray across the sample container 110.

Although not illustrated, a controller can be provided to control theoperation of the scanning system. The controller can be implemented tocontrol aspects of system operation such as, for example, focusing,stage movement, and imaging operations. In various implementations, thecontroller can be implemented using hardware, machine-readableinstructions or algorithm, or a combination of the foregoing. Forexample, in some implementations the controller can include one or moreCPUs or processors with associated memory. As another example, thecontroller can comprise hardware or other circuitry to control theoperation. For example, this circuitry can include one or more of thefollowing: field programmable gate array (FPGA), application specificintegrated circuit (ASIC), programmable logic device (PLD), complexprogrammable logic device (CPLD), a programmable logic array (PLA),programmable array logic (PAL) or other similar processing device orcircuitry. As yet another example, the controller can comprise acombination of this circuitry with one or more processors.

Generally, for focusing operations, a focus beam generated by a focusinglaser is reflected off of the sample location to measure the requiredfocus, and the sample stage is moved relative to the optical stage tofocus the optical stage onto a current sample location. The movement ofthe sample stage relative to the optical stage for focusing is generallydescribed as movement along the z-axis or in the z direction. The terms“z-axis” and “z direction” are intended to be used consistently withtheir use in the art of microscopy and imaging systems in general, inwhich the z-axis refers to the focal axis. Accordingly, a z-axistranslation results in increasing or decreasing the length of the focalaxis. A z-axis translation can be carried out, for example, by moving asample stage relative to an optical stage (e.g., by moving the samplestage or an optical element or both). As such, z-axis translation can becarried out by driving an objective lens, the optical stage, or thesample stage, or a combination of the foregoing, any of which can bedriven by actuating one or more servos or motors or other actuators thatare in functional communication with the objective lens or the samplestage or both. In various implementations, the actuators can beconfigured to tilt the sample stage relative to the optical stage to,for example, effectively level the sample container on a planeperpendicular to the optical imaging axis. Where this dynamic tilting isperformed to effectively level the sample locations on the samplecontainer, this can allow the sample container to be moved in the x andy directions for scanning with little or no movement in the z-axisrequired.

FIG. 2, which comprises FIGS. 2A and 2B illustrates an example opticalsystem for focus tracking. Particularly, FIG. 2A illustrates one exampleoptical design for focus tracking. FIG. 2B is a diagram illustrating analternative view of a portion of the optical system shown in FIG. 2A. Toavoid clutter and facilitate the reader's understanding, the exampleshown in FIG. 2A is illustrated with a single beam, which is a centerbeam in this case. The system may operate with more than one beam suchas, for example, with 3 beams. A three-beam system can provide, forexample, look-ahead and look-behind focus tracking.

Referring now to FIG. 2A, laser 270 generates light for the focusingbeams and is optically coupled into the system. Light from laser 270 canbe coupled via a fiber for example to a beam splitter prism 272, such asa lateral displacement beam splitter. Filters may be included, ifneeded, such as for source selection. Prism 272 splits the transmit beaminto two substantially parallel, spots of roughly equal intensity. Thiscan be included to provide for differential measurement in the focusingmodel.

A diffraction grating 274 generates multiple copies of the input beams.In other configurations, a beam splitter cube or multiple laser sourcescan be used to generate the multiple beams. In the case of a three-beamsystem, diffraction grating 274 may generate three output beams for eachof the two input beams. An example of this for one input beam is shownat FIG. 2B. Because the diffraction grating can generate beams that aredivergent (as also shown in FIG. 2B), a flat-top, or dove prism, 276redirects the multiple beams. In some implementations, the prism isconfigured such that the beams converge at the pupil of the objectivelens 142 so the beams at the sample container are normal to the samplecontainer. An example of this for a three-output-beam configuration isshown at FIG. 2B. The received signal from the sample container returnsthrough the beam splitter 277 and reflects off mirror 279. Because eachbeam pair is diverging, receive prisms 280 and 282 consolidate the spotsonto the focal plane of image sensor 284. In some examples, these can beimplemented as dove and roof prisms to refract and aim the rays exitingthe microscope object to fit on the image sensor array. A roof prism canbe used to refract the return beams to consolidate the spots within aspot pair onto the focal plane of the image sensor, and a dove prism torefract the fore/aft spot pairs to consolidate all spot pairs onto thefocal plane. With three-beam look-ahead, 3 beams pass through each ofthe two prism halves of the roof prism. However, in the other axis, thebeams are diverging, which is why the dove prism is included to correctthose.

In the various examples described above with reference to FIG. 2,various optical components are implemented using prisms. Some or all ofthese may be implemented using lenses, however prisms may be desirableas these components are generally less sensitive to misalignment ascompared to their lens counterparts. Prisms may also be more desirablethan lens systems because prisms are generally more compact and includefewer elements.

Objective lens 142 in the examples of FIGS. 1 and 2 provides a generallycircular field of view on the sample container. In one implementation,the center of the field of view is the current sample location beingimaged. The direction of scan within that field of view is either the xor the y axis. For purposes of discussion, the direction of scan will beassumed to be in the y direction. A light source, such as an LED orlaser light source generates the focusing beams. In the illustratedexample, three beams are used to provide a three-point differentialoff-axis predictive focus estimation—one beam for the current samplelocation and two additional beams for look-ahead and look-behind focustracking. These two additional beams are used to determine the focusdistance along the z axis between the optical stage and sample locationson the sample container.

The system described in FIGS. 1 and 2 illustrates an example system withwhich the systems and methods described herein may be implemented.Although the systems and methods may be described herein from time totime in the context of this example system, this is only one examplewith which these systems and methods might be implemented. The systemsand methods described herein can be implemented with this and otherscanners, microscopes and other imaging systems.

Pre-existing scanning systems use collimated light for focusingoperations. In such systems, collimated light, which maintains a fairlyconsistent diameter throughout the length of the beam, is directed tothe objective lens. An example of this is shown in FIG. 2A (describedabove), in which the collimated beams are delivered to the objectivelens. The objective lens focuses the collimated light onto the sample.Light reflected from the sample returns through the objective, and isre-collimated. The reflected, collimated beam is then directed to thesystem's image sensor (e.g., image sensor 284 in the example of FIG.2A). The reflected beams' locations on the image sensor are determinedfor focusing purposes. For example, with a two beam system the distancebetween spot locations on the image sensor is measured to determinefocusing.

While collimated light is a known light source and scanning systems, theinventors have discovered that collimated light can adversely affectfocus tracking operations in various applications. One adverse effectrelates to the spot size resulting from using collimated light for thefocus tracking beams. Because collimated light retains a relativelyconsistent diameter throughout the optical path the focus tracking beamsimage a relatively large spot size on the image sensor. The larger spotsizes encompass a greater number of pixels on the image sensor, whichincreases the number of rows of pixels of the image sensor that need tobe measured. This increases the amount of time required for focustracking operations.

Another adverse effect can arise in systems where the objective may beused to focus at multiple different surfaces but is not moved in anamount equal the distance between those different surfaces. In thisscenario, different spot sizes for the focus tracking beams reflectedoff the different surfaces can appear on the image sensor, impactingfocus tracking operations. FIG. 3 is a diagram illustrating an exampleof such a phenomenon. More particularly, FIG. 3 illustrates an examplein which a sample container containing one or more samples to be imagedcomprises multiple layers. With reference now to FIG. 3, samplecontainer 330 includes 3 layers 334, 338 and 336. In this 3-layerexample, there are four surfaces between the layers. These areillustrated at surfaces S1, S2, S3 and S4. Also illustrated in thisexample is an objective lens 332 which focuses the focus tracking beams333, 335 (there are 2 in a 2-beam system) onto sample container 330 forfocusing operations.

For focus tracking operations, it may be important to focus the imagingbeam to surface S2 in some instances and to surface S3 in otherinstances. Assume that the separation between the surfaces S2 and S3 isfixed at a distance X. In some applications, depending on the operationof objective lens 332, objective lens may move a distance greater thanor less than distance X when changing focus between surfaces S2 and S3.Consequently, focusing tracking beams 333, 335 reflected off surfaces S2and S3 may be re-collimated at a different diameter causing the S2 beamsto present a different spot size from the S3 beams.

An example of this is illustrated in FIG. 4. More particularly, FIG. 4illustrates a portion of an image sensor 362 comprising a plurality ofpixels (not shown for clarity of illustration) onto which the focustracking beams are directed. On the left-hand side of the figure inscenario 360, this illustrates an image sensor portion 362 with beamspots 34, 36 from each of the two focus tracking beams in a two-beamsystem. Spots 34 are from the left and right beams reflected off of oneof the two imaging surfaces (assume S2 in this example), and spots 36are from the left and right beams reflected off of the other of the twoimaging surfaces (assume S3 in this example). As illustrated in thisexample, based on the movement of the objective lens, the two focustracking beams, which are both collimated and which both havesubstantially the same beam diameter before entering the objective lens,now have different diameters and therefore produce different spot sizeson the image sensor. The larger two of the spots each encompass agreater number of pixels and therefore increases the number of rows ofpixels of the image sensor that need to be measured. This increases theamount of time required for focus tracking operations. For thesereasons, it is desired to achieve a scenario such as scenario 361illustrated on the right-hand side of FIG. 4 in which the spots 34, 36from the left and right beams reflected off of surfaces S2 and S3,respectively, are substantially the same spot size and are relativelysmall.

Pre-existing systems may use a focusing lens to cause the focus trackingbeams to converge on the image sensor and to reduce or minimize theirspot sizes on the sensor. However because a lens introduces a curvedsurface in the optical system, slight changes in alignment of the lens,including changes that can arise through thermal variations in thesystem, can cause inaccuracies in the placement of the focus trackingbeams on the sensor. Movement or changes in the lens can cause a lateraltranslation that affects the multiple focus tracking beams differently.Accordingly, as described above with reference to FIG. 2, in someexamples the focusing lens is replaced with a roof prism.

FIG. 5A is a diagram illustrating an example of a focusing lensimplemented to cause to focus tracking beams to converge onto an imagesensor. With reference now to FIG. 5A, light from a light source (e.g.the laser diode 270 of FIG. 2) is delivered by a fiber (laser and fiberare not shown) to a collimating lens 400. The collimated light is splitinto two beams such as by a beam splitter prism 382 (e.g., beam splitterprism 272 of FIG. 2). To avoid unnecessary clutter in the illustrationtwo reflected focus tracking beams 394, 395 are illustrated at the lens370 and image sensor 398; however, only one of the two focus trackingbeams is illustrated in the remaining portions of FIG. 5A.

The focus tracking beams from beam splitter prism 382 pass through beamsplitter 384 and are reflected by mirror 386 through objective lens 390.Objective lens focuses the beams onto the sample in the sample container392 (e.g., sample container 330 of FIG. 3). In this example, the focustracking beams is reflected off of the S2 surface from sample container392. The reflected beams (still only one beam 394 illustrated) returnsthrough objective lens 390, are reflected off of mirror 386 and beamsplitter 384, and are directed toward lens 370. Because return beams394, 395 are diverging at this point, lens 370 is implemented to causethe return beams 394, 395 converge on to the image sensor 398. Also,because the focus tracking beams 394, 395 are collimated light, lens 370serves the additional function of focusing the beams into a smaller spotsize on image sensor 398. However because changes in the lateralplacement of lens 370 affect the positioning of the beams on imagesensor 398, these changes introduce focus tracking error.

FIG. 5B is a diagram illustrating an example in which lens 370 isreplaced with a roof prism 396 to avoid issues caused by changes in thelateral placement of lens 370. Replacing the lens with a roof prism 396reduces or eliminates sensitivity of the system to the lateral positionof the lens. Changes of the prism due to thermal and other variations donot impact the spacing of the focus tracking beams 394, 395 on imagesensor 398. Because the angular deviation of a prism is completelydetermined by the angle of the glass, lateral displacement of the roofprism 396 does not affect the beams.

The inclusion of a roof prism 396 in place of a lens 370 can improve theaccuracy of the focus tracking system. Because the separation betweenthe spots is used to measure distance from the objective lens to thesample container, higher levels of accuracy are achieved when theseparation of the beams is dependent only on the distance to the samplecontainer. Other variables that affect the beam separation, such asthose introduced by lateral imprecision in placement of lens 370,negatively impact the accuracy of the focus tracking system.Accordingly, including a roof prism, which presents the same angle tothe focus tracking beams even in the presence of some lateraldisplacement, can greatly benefit the system's accuracy.

There is a drawback to removing the lens. Because the lens iseliminated, the focus tracking beams (beams 394, 395 in this example)are not focused on the sensor. Therefore, in various examples, ratherthan use collimated light as is done with pre-existing scanning systems,the focus tracking beams are focused to place a waist at a given pointalong the optical path. This presents a smaller spot size on the imagesensor. For example, in one application, collimating lens 400 is movedfarther away from the fiber output than it would otherwise be placed tocollimate the light from the fiber. The point along the optical path atwhich collimating lens 400 is placed dictates the position at which beamwaist is placed along the optical path. Collimating lens 400 can bepositioned to provide a waist such that, despite replacing the lens 370with roof prism 398, the reflected focus tracking beams 394, 395 can befocused on to the image sensor 398 with a reduced spot size.

Another benefit of moving collimating lens 400 to place a beam waist inthe optical path is that this may help to reduce or eliminate animbalance in spot size that was discussed above with reference to FIG.4A. Lens 400 can be provided and positioned in the optical path suchthat the light returned from the sample, through the objective lens, andthrough the remainder of the optical path, impinges on the sensor withsubstantially the same spot size as illustrated in scenario 361. Moreparticularly, in some instances a lens is positioned a distance from thefiber output to place a beam waist at a predetermined distance away fromthe collimator to balance the diameters of the beams that propagate fromthe upper and lower surfaces of the sample container want to the imagesensor.

In one application, the beam waist is positioned at a distance of 690mm-700 mm away from the collimator to balance and reduce the diametersof the beams impinging on the image sensor. In some examples, the spotsize can be reduced to approximately 400 μm. In other examples, spotsize can be in the range of 300 μm to 500 μm. In yet other examples,other spot sizes can be used.

Additionally, movement of collimating lens 400 to place a beam waist inthe optical path can help to balance the intensities of the lightimpinging on the image sensor. FIG. 4B is a diagram illustratingintensities of left and right focus beams reflecting onto the imagesensor from the S2 and S3 surfaces at different focus settings with thecollimating lens adjusted to provide a beam waist at a non-optimallocation. In this diagram, spot brightness is on the vertical axis andthe position of the focusing stage is on the horizontal axis. Thevertical blue line on the left-hand side of the diagram illustrates anoptimal focusing position for the S2 reflections in one exampleimplementation. Similarly, the vertical blue line on the right-hand sideof the diagram illustrates an optimal focusing position for the S3reflections in this example implementation. As this diagram illustrates,the average spot brightness for the S2 beams is approximately 170 at theS2 focusing position, while the average spot brightness for the S3 beamsis approximately 85 at the optimal S3 focusing position. Accordingly,spot intensity for the S2 and S3 beams is not balanced.

FIG. 4C is a diagram illustrating intensities of the left and rightfocus beams reflecting onto the image sensor from the S2 and S3 surfacesat different focus settings with the collimating lens adjusted toposition at the beam waist more optimally along the optical path of thefocus tracking beams. Here, with a beam waist is positioned along theoptical path, the intensities of the S2 and S3 reflected beams are morebalanced. Particularly, the diagram illustrates that the left and rightS2 beams have an average spot brightness of approximately 125 at the S2best focus position. This also illustrates the left and right S3 beamshave an average spot brightness of approximately 130 at the S3 bestfocus position. As a comparison of FIGS. 4B and 4C illustrates,placement of a beam waist along the optical path can affect the balanceof intensities of the S2 and S3 focus tracking beams.

FIG. 6 is a diagram illustrating an example configuration including alens positioned to place a beam waist of the focus tracking beam at aselect position. In this example, light from a light source (notillustrated), such as a laser light source (e.g., light source 270), iscarried by fiber-optic cable 432 which is connected to a lens housingassembly via a ferrule 434. Ferrule 434 is mounted in a mounting block435 that is fixedly attached to insert 436. Lens 440 of a given focallength is placed at a determined distance from the output of fiber 432,and can be maintained at this distance by the lens housing assembly. Inthis example, light from the fiber travels through an aperture in aninsert 436 mounted in body portion 438. The focal length of the lens 440and its distance from the output of fiber 432 are chosen to place thebeam waist at the desired position along the optical path. As notedabove, the distance between the output of the fiber and lens 440 ischosen to place the beam waist at the desired position, as describedmore fully below.

In this example, separation between lens 440 and the fiber output is14.23 mm, which is the working distance between the lens surface and thefiber. 15.7 mm is the effective focal length of the lens (which ishigher than the back focal length of the lens because it is in respectto the lens principal plane). Because the back focal length of the lensin the collimator is 13.98 mm, which is the distance from the lensvertex to the focal point of the lens on the optical axis, the backfocal length is shorter than 14.23 mm.

In the illustrated example, insert 436 is slidably mounted within acavity defined by body portion 438 such that the distance between thefiber output and lens for 40 can be adjusted by insert 436 slidablymounted within the cavity of body portion 438. A set screw 442 or otherlocking mechanism can be included to lock insert 436 into place withinbody portion 438. Use of a slidable insert 436 allows the system to beadjusted to tune or optimize the spot size on the image sensor. This canallow for final system configuration adjustments or in-field tuning. Theexample illustrated in FIG. 6, lens 440 is a plano-convex lens. However,after reading this description, one of ordinary skill in the art willunderstand that other lens structures can be used, including, forexample, a biconvex lens.

In some applications, the lens is configured such that the beam waist ispositioned within the objective lens. More particularly, in someapplications, the lens is configured such that the beam waist ispositioned within the objective lens before the beam impinges on thesample while in other applications the lens is configured such that thebeam waist is positioned within the objective lens after the beam isreflected off the sample. In other applications, the lens is configuredsuch that the beam waist occurs before the objective lens, after thereflected beam leaves the objective lens or between the objective lensand the sample. Placement of the lens can be determined by an iterativeprocess, such as through the use of modeling software, to achieve thedesired spot size and balance on the image sensor.

In addition to balancing the spot sizes, smaller spot sizes aregenerally utilized to improve the speed with which focusing can bedetermined. The time required to read information from the image sensoraffects the latency of the focus tracking system. More particularly, fora sensor with a given pixel density, a larger spot size covers morepixels and more time is required to read the data from each pixel withinthe spot diameter. Accordingly, as discussed above the lens used tobalance the beam diameters can also be used to reduce the size of thespot impinging on the image sensor, thereby reducing the amount of timerequired to determine the spot location (or locations for multiple beamfocusing) for focusing operations.

As discussed above with reference to FIG. 3A, in some applications, amultilayer sample container can be used to carry the sample to be imagedby the scanning system. As discussed in that example, the sample to beimaged may be contained in solution in layer 338. In order for imagingto occur, at least layer 334 must be optically transparent to the beamused for imaging. Layer 336 may be optically transparent as well.Accordingly, surfaces S1, S2, S3 and S4 are generally reflective.Likewise, because it is important for the imaging beam to reach thesample at layer 338, it is undesirable to use antireflective coating onthe surfaces. Accordingly, unwanted reflections from surfaces S1 and S4during focus tracking and imaging operations can create unwanted opticalnoise in the system and can obscure the reflected beams from S2 and S3,which are the beams to be collected at the image sensor.

FIG. 3B is a diagram illustrating an example of the creation of unwantedreflections off of the multiple surfaces of a multilayer samplecontainer in some environments. As seen in this example, a three-layersample container includes surfaces S1, S2, S3 and S4. For clarity, asingle focus tracking beam 465 is illustrated. However, in otherapplications, multiple focus tracking beams can be used. For instance,examples below describe a system in which two focus tracking beams aredescribed. As also seen in this example, a beam is reflected off each ofthe surfaces S1, S2, S3 and S4. Because the sample is between surfacesS2 and S3, those are the surfaces on which the system is designed tofocus. Accordingly, the reflected beam 467 off of surface S1 and thereflected beam 469 off of surface S4 do not return any usefulinformation and are unwanted reflections. The reflections of interestfor focus tracking are reflections off of surfaces S2 and S3.Accordingly, if light from the reflections off of surfaces S1 and S4were to reach the detector, this could introduce noise that couldinterfere with detection of the focus tracking beam reflections.

FIG. 3C is a diagram illustrating an example of the effect of theunwanted reflections on the image sensor. As seen in this example, inaddition to the spots 482 presented by the focus tracking beams, thereis a significant amount of noise appearing on the image sensor as aresult of the unwanted reflections. In other examples, the unwantedreflections can also appear as additional spots on the image sensor.FIG. 3D is a diagram illustrating a reduction in noise at the imagesensor as a result of the placement of blocking structures in accordancewith examples discussed below.

This problem is exacerbated in circumstances in which the reflectionsoff of surfaces S1 and S4 are of greater intensity than the reflectionsoff of the sample. Because it is important for the sample container tobe optically transparent, antireflective coatings are not provided onthe sample container. Likewise, reflections off a glass surface tend tobe stronger than reflections off of a biological sample. Additionally,in applications in which the sample container contains a nano-well orother like pattern on surfaces S2 and S3, this can further diminish thereflections off of those surfaces. Accordingly, the unwanted reflectionsfrom surfaces S1 and S4 tend to be of greater intensity than thereflections off of surfaces S2 and S3. For example, in some applicationsthe reflections off of surface S1 can be as much as 100 times (orgreater) the intensity of the reflections off of surfaces S2 and S3. Toremedy this problem and remove the impact of these unwanted reflectionsfrom the focus tracking operations, various examples may be implementedto include blocking structures at determined locations along the opticalpath between the sample and the image sensor to block this unwantedlight from reaching the image sensor.

FIG. 7 is a diagram illustrating another example of the scanning systemwith which examples of the systems and methods described herein may beimplemented. With reference now to FIG. 7, this example includes a lightsource (not illustrated), such as a laser light source. For example, inone application, the light source can be configured as a laser diodecoupled to the system using a fiber coupler and a lens structure such asthe example illustrated in FIG. 6. As another example, the light sourcecan be configured as a laser diode with a collimator to providecollimated light for focus tracking operations.

In this example, light from the laser is introduced into a lateraldisplacement prism 522 to separate the light into two parallel beams.Other configurations may be implemented with a single focus trackingbeam or with more than two focus tracking beams. In operation, the focustracking beams are sent through beam splitter 524 and are reflected offof upper periscope mirror 526 and lower periscope mirror 528. The focustracking beams are delivered through periscope window 530 and beamsplitter 532 (which may also be implemented as a dichroic filter). Thebeams are then reflected off of mirror 536 and focused by objective lens538 onto the sample container 540. Reflections from the sample containerare returned through the objective lens and follow the same path untilthey are reflected off of beam splitter 524. Because the beams may bediverging from one another slightly, a roof prism 546 may be included toredirect the beams to a parallel orientation or even to a slightlyconverging configuration such that they can both be directed toward arelatively small area image sensor. In this example, camera turningmirror 548 directs the focus tracking beams to the image sensor 550.Although the example blocking structures described herein are describedin terms of this example configuration, one of ordinary skill in the artafter reading this description will appreciate how different geometriesor placement of blocking structures can be used in differentlyconfigured systems to block unwanted reflections from a multi-surfacesample container.

The example system of FIG. 7 was modeled to determine the paths of thebeams reflected off of the S1-S4 surfaces in the system to identifypoints along the return path at which the unwanted reflections from theS1 and S4 surfaces could be blocked from reaching the image sensor. Thespatial relationship of the beams at various points along the path as aresult of this modeling is illustrated at FIGS. 8, 9, 11, 12, 19, 20,21, 22, 23 and 24. As these figures illustrate, the spatialrelationships of the beams reflected off of surfaces S1-S4 variesthroughout the return path of the system. The beam locations arechanging relative to one another along the length of their return path,and the locations also change depending on placement of the samplecontainer in respect to the objective. Adding to the complexity is thatthere are focus beams running in the forward and return directions andthere are also imaging beams running in both directions as well.Therefore, it is not a trivial task to configure in place blockingstructures within the optical path that effectively block unwantedreflections from imparting noise on the image sensor while avoidinginterference with the desired focus tracking and imaging beams.

FIGS. 8 and 9 are diagrams illustrating the spatial relationship ofreflected focus tracking beams at beam splitter 532 in the exampleconfiguration of FIG. 7 using a multi-layer sample container such asthat illustrated in FIG. 3B. FIGS. 8 and 9 show the beams within a 21mm×21 mm area. FIG. 8 shows the spatial relationship of the beams atbeam splitter 532 when the system is configured to focus on the top ofthe sample well at surface S2, while FIG. 9 shows the spatialrelationship of the beams at beam splitter 532 with the systemconfigured to focus on the bottom of the sample well at surface S3.These figures illustrate that at beam splitter 532 the reflected beams,with the system focused at S2 and S3, impinge on the surface in threespatial groups: the reflection of the left focus tracking beam off ofsurfaces S1, S2 and S3 are in a first group; the reflection of the rightfocus tracking beam off of surfaces S1, S2 and S3 are in a second groupthat is physically separated from the first group; and that left andright focus tracking beams reflected off of surface S4 are in the areain between these two groups. With this spatial relationship among thebeams, it would be difficult to use an aperture configuration toeffectively block the left and right reflections off of surface S1 whileallowing the desired reflections off of surfaces S2 and S3 to passuninhibited. However, because there is good spatial separation of thereflections off of surface S4 relative to the other reflections, thereflections from the S4 surface may be blocked at this point along thereturn path.

FIG. 10 is a diagram illustrating an example placement of a beam blockerto block reflections of the left and right focus tracking beams from theS4 surface in accordance with one example implementation. This exampleshows the reflections 424 from surface S4 converging with one another atbeam splitter 532 as was seen in FIGS. 8 and 9. This example alsoillustrates how a blocking structure can be included to block thesereflections from surface S4 without interfering with the desiredreflections from the S2 and S3 surfaces. This can be implemented in theillustrated example using a 4 mm wide obscuration on the focus trackingmodule side of beam splitter 532.

FIGS. 11 and 12 are diagrams illustrating the spatial relationship ofreflected focus tracking beams at beam splitter 532 in the exampleconfiguration of FIG. 7 using a multi-layer sample container such asthat illustrated in FIG. 3B. FIGS. 11 and 12 show the beams within a 25mm×25 mm area. FIG. 11 shows the spatial relationship of the beams attop periscope mirror 526 when the system is configured to focus on thetop of the sample well at surface S2, while FIG. 12 shows the spatialrelationship of the beams at top periscope mirror 526 with the systemconfigured to focus on the bottom of the sample well at surface S3.Because reflections of the focus tracking beam off of the S4 surface inthis example are blocked at beam splitter 532 before reaching this pointin the return path, there are no spots from surface S4. Moreimportantly, this shows that the reflected beams from surface S1 havegood spatial separation from the desired reflections off of the S2 andS3 surfaces.

With this spatial placement of the beams, an aperture can be used toblock the S1 reflections while allowing the reflected beams from the S2and S3 surfaces to pass through and ultimately reach the image sensor.FIGS. 13 and 14 illustrate the beams reflected off of top periscopemirror 526 and beam splitter 524. As this illustrates, if the beams werenot blocked at top periscope mirror 526, they would reflect off of beamsplitter 524 and impinge on the edges of roof prism 546. As thismodeling illustrates, reflected beams from surface S1 can be blocked byplacing a 20 mm×20 mm aperture at upper periscope mirror 526.Alternatively, the size of upper periscope mirror 526 can be reduced toa 20 mm×20 mm dimensions so that the reflected beams from the S1 surfaceare not returned to the image sensor. In other applications or for otherplacement locations for the aperture, the size of the apertureimplemented can vary based on the position of the S1 beams. In anotherexample implementation, the aperture is 20.8 mm wide. This width waschosen to accommodate an S2 image at about −20 μm to +30 μm (about bestfocus for S2 in one application) and an S3 image at about −25 μm to +25μm (about best focus for S3 in one application).

FIG. 15A provides a top-down view illustrating the focus tracking beamsreflected from the sample through objective lens 538 and directed towardbeam splitter 532. Although mirror 536 is not shown in FIG. 15A, thisillustrates the reflected focus tracking beams being reflected towardbeam splitter 532. This example also illustrates the S4 reflected beamsbeing blocked by a beam blocker positioned at the back face of beamsplitter 532. Although the beam blocker is not illustrated in FIG. 15A,one illustrative example is provided in FIGS. 16A and 16B.

FIG. 15B provides a close up view of FIG. 15A, illustrating an exampleof the focus tracking beams reflected from surface S4 at the rear faceof beam splitter 532. As this example illustrates, the focus trackingbeams reflected from surface S4 are blocked by a blocking member 562. Asthis example also illustrates, the front face of blocking member 562 isoriented to be substantially parallel with the back face of beamsplitter 532. In one example implementation, blocking member 562 isdisposed in the system to be separated from the back face of beamsplitter 532 by 50 μm. In other examples, other separation spacings canbe provided. For example, in some implementations, the spacing can be inthe range of 25 μm-100 μm. Although this example illustrates blockingmember 562 as having a rectangular cross-section, blocking member 562can be implemented using other shapes or geometries, an example of whichis illustrated below with reference to FIGS. 16A and 16B.

FIG. 15C is a diagram illustrating a top-down view of an example of ablocking member and splitter positioned within a portion of an imagingsystem. In this example, blocking member 562 is positioned at the rearface of beam splitter 532 to block beams reflected from the S4 surface.The reflected beams emerging from objective lens 538 are reflected bymirror 526 toward beam splitter 532. Blocking member 562 is positionedto block the beams reflected from the S4 surface and is of asufficiently small width so as to not interfere with the beams reflectedfrom the S2 and S3 surfaces.

In the illustrated example, blocking member 562 is 4 mm wide and 2 mm inlength, and it is slightly offset from the optical axis of objectivelens 538. It is, however, aligned with the center of lower periscopemirror 528, which is mounted in housing 565. More particularly, in oneexample implementation, blocking member 562 is offset 1.1 millimeters tothe left of the objective optical axis to ensure that it is centeredrelative to the beams reflected from the S4 surface.

FIG. 15D is a diagram illustrating a representation of a 4 mm wideblocking structure in the beam path of the reflected focus trackingbeams at the splitter. As this example illustrates, a 4 mm wide blockingstructure (represented by rectangle 631) is of sufficient width to blockthe focus tracking beams reflected from surface S4, which are shown inthe center of the diagram. As this example also illustrates, the widthof the blocking member is chosen to be wide enough to block the unwantedreflected beams, but still provide the largest possible capture rangesfor S2 and S3 imaging. Because slight changes in the focusing can have acorresponding change in the position of the beams at the splitter, thewidth of the blocking member can be chosen as being somewhat wider thanthat which would be necessary to block the beams in a perfect focuscondition. In other words, the blocking member can be wide enough toaccommodate a determined degree of imprecision in the focusing system.

FIGS. 16A and 16B are diagrams illustrating an example of a beam blockerthat can be used to block the S4 reflections at beam splitter 532 inaccordance with the example implementations described with reference toFIGS. 8-10. FIGS. 17 and 18 are diagrams illustrating an exampleplacement of the beam blocker illustrated in FIGS. 16A and 16B. Theleft-hand side of FIG. 16A illustrates a rear view (from the perspectiveof the beam) of beam blocker 620; and the right-hand side illustrates aperspective view of beam blocker 620. Beam blocker 620 includes a frameportion 622 defining an aperture 624 through which the reflected beamscan pass. A beam blocking member 626, which includes a blocking face630, is supported in position by extension arms 628 to block theunwanted reflection beams from S4. In the illustrated example, extensionarms 628 are elongate structural members attached, affixed, joined orotherwise connected to opposite sides of frame portion 622, and beamblocking member 626 extends across the distal ends of extension arms628.

Frame portion 622 and extension arms 628 provide a mounting structure bywhich the beam blocking member 626 can be mounted in position at beamsplitter 532 without interfering with reflections from surfaces S2 andS3. Beam blocker 620 can be cast, molded, machined or otherwisefabricated as a unitary structure. In other examples, the elements thatmake up beam blocker 620 can be separate components that are attached,joined, fastened or otherwise connected together to form the resultingassembly. Beam blocker 620 can be implemented using light-absorbing,opaque surfaces to avoid other unwanted reflections within the system.For example, beam blocker 620 can be made using black anodized aluminumor other light-absorbing, or light-absorbing-coated materials. Beamblocker 620 can be dimensioned for a particular application. In oneexample application, beam blocker 620 is dimensioned to provide: anaperture width of 30 mm and a height of 21 mm; extension arms 628 of 25mm in length; and a blocking surface that is 2.8 mm wide and 21 mm inlength.

With reference now to FIG. 16B, view 682 illustrates a top-down view ofbeam blocker 620, and view 683 illustrates a cross-sectional side viewat A of beam blocker 620. The front edge of extension arms 628 istapered to conform to the angle of beam splitter 532 as furtherillustrated in FIG. 17 (described below). The beam blocking member has atriangular cross-section and is oriented to present flat blocking face630 to the incoming beam. Although beam blocker 620 can be made usinglight-absorbing materials, presenting a triangular cross-section to theunwanted beams can have the effect of reflecting any unabsorbed lightout of the return path.

FIG. 17A presents a cutaway view of beam blocker 620 installed at beamsplitter 532. With reference now to FIG. 17A, in operation, reflectionsof the focus tracking beams from surfaces S1, S2, S3 and S4 travel upfrom the objective lens, are reflected off of mirror 536 and directedtoward beam splitter 532. Blocking face 630 (see FIGS. 16A and 16B) ofblocking member 626 blocks the S4 reflections from continuing past beamsplitter 532. It is shown in this example that extension arm 628 isdimensioned so as to place blocking member 626 at or near the surface ofbeam splitter 532. This figure also illustrates the tapered front angleof extension arm 628 to allow the blocking face 630 of blocking member626 to be placed adjacent to and at substantially the same angle as beamsplitter 532. In some examples, blocking member 626 is positioned suchthat blocking face 630 is in touching relation with beam splitter 532.In other examples, blocking member 626 is positioned such that blockingface 630 is separated from the face of beam splitter 532 by a smallamount such as, for example, by 50 μm to 500 μm.

In alternative examples, a blocking element can be disposed on the backside of beam splitter 532 without the structure illustrated in FIGS. 16and 17. For example, in some instances, a strip of opaque material canbe attached to the rear surface of beam splitter 532. In otherinstances, an opaque or optically absorbent coding can be applied in anarrow stripe to the back of beam splitter 532.

For scanning operations, the imaging beams, which for example can be redand green imaging beams, enter the system from the right-hand side asillustrated by arrow 690. These beams are reflected off the front faceof beam splitter 532 toward mirror 536. Mirror 536 reflects the imagingbeams downward into the objective lens. Accordingly, the position ofblocking member 626 is also selected so as not to interfere with theimaging beams reflected toward the sample (by front surface of beamsplitter 532).

This example also illustrates that blocking member 626 presents atriangular cross-section, with the rear edges of blocking member 626tapering to meet at an acute angle. Other cross-sectional geometries forblocking member 626 can be used, provided that blocking face 630 isproperly dimensioned to block or substantially block reflections fromsurface S4. However, a geometry such as that illustrated, which reducesthe cross-section toward the rear of blocking member 626 can minimizethe chance that blocking member 626 may otherwise provide unwantedinterference with desired beams.

FIG. 17B presents a rear view of beam blocker 620 installed at beamsplitter 532. This illustrates the frame portion 622 mounted in placeusing bolts 732. This illustrates the window provided by aperture 624that allows the light reflected from surfaces S2 and S3 (and S1, whichis blocked later in the path) to pass, while blocking member 626 blocksthe light from surface S4 before as it leaves beam splitter 532.

FIG. 18A illustrates an example of an aperture that can be used to blockthe beams reflected off the S1 surface. In one example, this can beplaced on the inside wall of the focus tracking module at the periscopeaperture. As noted above, in one example implementation the aperture is20.8 mm×20.8 mm, but in other examples other aperture sizes can beprovided. As with the blocking member, the dimensions of the aperturecan be chosen to block the unwanted reflections while providing thelargest capture range possible for the S2 and S3 reflected beamsrelative to “best focus” considerations. FIG. 18B illustrates an exampleplacement of the aperture 740 in front of the beam splitter 524 normalto the beam axis.

FIGS. 19 and 20 show the results of the addition beam blocker 620 toblock S4 reflections and a 20.8 mm×20.8 mm aperture to block S1reflections. FIG. 19 shows spots from the beams at top periscope mirror526 for focusing at the top of the sample (surface S2), and FIG. 20shows spots from the beams at top periscope mirror 526 for focusing atthe bottom of the sample (surface S3).

Although the foregoing was illustrated with objective focusing atsurfaces S2 and S3, perfect focusing is not always achieved andtherefore examples can be implemented to account for a capture rangeabove and below the upper and lower sample. For example, the abovemodeling was also carried out assuming a “best focus” that accommodatesfocusing within +/−25 μm from the upper and lower sample surfaces. This“best focus” modeling confirmed that the above described structures aresufficient to block unwanted reflections from the S1 and S4 surfacesunder best-focus operations.

FIGS. 21-24 are diagrams illustrating spot placement at the image sensorat the top and bottom of an example “best focus” capture range. In thisinstance, modeling was performed with the capture range of +/−25 μm.These diagrams show an image sensor area of 11.26 mm×11.26 mm. FIG. 21illustrates spots at the camera for the S2, S3 reflected beams forimaging at the top of the capture range for focusing on S2 with theobjective position 1.064 mm from S2. FIG. 22 illustrates spots at thecamera for the S2, S3 reflected beams for imaging at the bottom of thecapture range for focusing on S2 with the objective position 1.014 mmfrom S2. FIGS. 21 and 22 illustrate +/−25 μm variation from ideal focusposition. FIG. 23 illustrates spots at the camera for the S2, S3reflected beams for imaging at the top of the capture range whenfocusing on S3. FIG. 24 illustrates spots at the camera for the S2, S3reflected beams for imaging at the bottom of the capture range whenfocusing on S3.

As described above, in focus tracking operations with the multi-beamsystem, spot separation, or the distance between the spots of the focustracking beams on the image sensor is measured to determine focusing.Accordingly, stability of the spot separation can be an important factorin achieving accurate measurements. Spot separation stability can beimpacted by factors such as movement of the focusing stage (sometimesreferred to as the Z stage) spot quality/shape as a function of time,and resolution of the centroid algorithm used to resolve the spotseparation. One challenge with spot separation stability is that spotsinherently include fringes. Due to mode hopping of the laser, fringepatterns can change, which induces a variation in spot profile over timethat affects the focus tracking module's spot separation stability. Anexample of this is illustrated in FIG. 25A, which illustrates spotfringe variation. This example shows spot fringe variation for laseroperated at a power of 12 mW with the exposure time of 250 μs, with anOD 1.0 ND filter in place.

Operating a laser in a mode commonly referred to as AmplifiedSpontaneous Emission (ASE) tends to provide a cleaner spot profile. Anexample of this is illustrated in FIG. 25B. This example is for the samelaser diode operated at 500 μW, 250 us exposure (No ND filter). In thismode the source emits temporally incoherent light, behaving more like anLED rather than a laser and has a wide optical bandwidth of 5 to 10 nmFWHM (full width at half maximum intensity). However, there are severaldisadvantages to operating in ASE mode, which is why typicalpre-existing imaging systems are not operated in such a mode. First, ASEmode is not a lasing mode for the laser diode, therefore the outputpower is very low. It is generally defined as a mode below the lasingthreshold in which no lasing occurs. As such, its output is temporallyincoherent, and includes frequency components across a broad-spectrum.

FIG. 26 is a diagram illustrating an example of a laser diode operatedin an ASE mode. In this example, the laser diode is operated at 0.17 mWand exhibits a relatively flat spectrum (when compared to the diodeoperating in a lasing mode) with frequency components across a broadrange of wavelengths. There is no single mode of operation and theoutput is not coherent. Incoherence in the light source can lead toundesirable effects such as destructive interference and chromaticaberrations. Additionally, it may simply be impractical to operate in anASE mode because there is not sufficient power emitted to produce a beamof sufficient intensity. There are other applications, however, in whicha laser may be operated in ASE mode. In this mode, the laser diode tendsto act more like an LED and, as such, it may be useful for certainapplications.

FIG. 27 is a diagram illustrating an example of the same laser diodeoperated in a lasing mode. The diagram the top half of FIG. 27illustrates the same laser diode operated at 0.96 mW, and the diagram inthe bottom half of FIG. 27 illustrates the same laser diode operating at1.22 mW. In both cases, the output is highly coherent with effectively asingle dominant peak at the operating frequency and almost negligentsecondary peaks. This is in stark contrast to the ASE mode which did nothave a dominant peak.

FIG. 28 is a diagram illustrating an example of a laser diode operatedin a hybrid mode. FIG. 28 shows the laser in this example operating at0.43 mW. At this power level a few dominant peaks are beginning to formbut there are still strong secondary peaks. As this diagram illustrates,the laser diode is not in a strong lasing mode, yet it is also not in afull ASE mode. Power level levels may still be defined as above thelasing threshold, but the output is not fully coherent.

Because the ASE mode may produce output without enough power, operationin the ASE mode is not operationally practical. As noted above withreference to FIG. 25A, however, operating the scanning system in lasingmode creates temporally variant fringes which provides instability inspot measurement.

An example of this is illustrated at FIG. 29, which shows instability inthe spot's morphology when the laser diode is powered to operate in alasing mode in accordance with one example of the systems and methodsdescribed herein. As shown in this figure, the standard deviation of theleft beam spot on the image sensor is 1.619 pixels and the standarddeviation of the right beam spot on the image sensor is 0.518 pixels.However, as the graphs for the left and right spots illustrate, motionof the spot for each beam can be dramatic from one frame to the next,and indeed can shift several pixels. The beam profile for two adjacentframes of the left spot is shown in the profile images on the right-handside of the figure. These profile images illustrate how the deviation ofbeam spot placement arises over time.

Because focus is determined by measuring the distance between the leftand right spots on the image sensor, variations in spot placement canlead to inaccuracies in focus tracking. The impact of the movement ofthe left and right beams as shown in the top two graphs of FIG. 29 isillustrated in the bottom graph of the figure. This graph shows thechange in distance, referred to here as Delta X, between the left andright spots over the same number of frames. This shows a standarddeviation of 1.178 pixels, which leads to a spot separation stability of+/−139 nm with 95% confidence interval (^(˜)2*StDev for a Gaussianpopulation). This is calculated as shown in the figure as(1.178*1.96)/16.36=+/−139 nm. The 16.36 factor represents the Focustracking Gain in pixels/μm. It represents how many pixels of spotseparation are obtained for every 1 μm shift of objective-to-sampledistance. It is used for the conversion of a delta in spot separation(pixels) to a delta in space in z direction (nm).

The inventors have discovered that the interference fringe patternsarise due to the multilevel structure of the sample container as shownin FIG. 3A. The inventors have further discovered that this is a resultof the superimposition of multiple beams and/or scattered light withinthe multi-layer glass sample container. Changes in the position of thesample container (e.g., in the X and Y direction) with no other changescan result in movement of the fringes.

FIGS. 30A and 30B illustrate additional examples of spot movement withthe laser diode operating in a hybrid mode. Particularly, FIGS. 30A and30B illustrate a more optimal scenario with a stable laser that is notmode hopping. As illustrated in FIG. 30A, standard deviation of the leftspot is down to 0.069 pixels, and the right spot is down to 0.064pixels. As the upper two graphs on the figure indicate, spot movementfrom frame to frame is generally less than one pixel. Because themovement can be additive, the Delta X difference between the left andright spots can have a standard deviation of 0.122 pixels. This reducesthe spot separation stability to +/−15.2 nm ((0.122*1.96)/16.36nm=+/−15.2 nm). Here, 16.36 is the FTM gain in pixel/μm. This is theamount of Delta X in pixel that is obtained when the objective moves 1μm in the Z axis. This can be used to covert from pixels in Delta X toμm in the Z space and vice versa. Additionally, 1.96 is themultiplication factor for the standard deviation to express 95%confidence interval of the error of the distribution (assuming it's aGaussian distribution).

In the example of FIG. 30B, the standard deviation of the left spot isdown to 0.069 pixels, and the right spot is also down to 0.069 pixels.As the upper two graphs on the figure indicate, spot movement from frameto frame is generally less than one pixel. Because the movement can beadditive, the Delta X difference between the left and right spots canhave a standard deviation of 0.127 pixels. This reduces the spotseparation stability to +/−14.6 nm ((0.127*1.96)/16.36 nm=+/−14.6 nm).

As noted above, it is impractical to run the laser in a pre-existing ASEmode. As also just described, accuracy suffers with the laser dioderunning at a power level above the lasing threshold, and this isespecially true if mode hopping occurs (such as, for example, via powervariations). However, the inventors have discovered that operating thelaser in a hybrid mode, between the ASE mode and a full lasing mode,provide sufficient beam intensity for measurement at the image sensorand increased spot placement stability for improved measurementaccuracy. This mode can be achieved in some instances by operatingslightly above the lasing threshold of the laser diode. For example,this might occur slightly past the knee of the lasing curve, but isstill low enough that a significant portion of the power is in the ASEstate. This produces an output where a large amount of the light stillhas a broader spectral width resulting in significantly reducedcoherence.

Operating a laser in this hybrid mode can be advantageous as compared toother light sources that might be used to attempt to achieve the sameeffect. Laser diodes tend to be desirable light sources because theyexhibit a high reliability and low cost, due to the high volumemanufacturing of this type of devices by different companies in thefield. Operating laser diode in this lower power mode can even increasethe typical high MTBF ratings that can be achieved by laser diodes.Therefore it is possible to achieve the result of a device with veryhigh lifetime and MTBF rating (combination of the laser diodecharacteristics and very low operating power), low cost of manufactureand short enough coherence length to eliminate interference fringescaused by the sample container's multi-layer structure.

Table 1 is a diagram illustrating spot separation stability with variousalternative solutions implemented. The first group of measurementsassumes a laser power of 12 mW to operate in a lasing mode, presence ofan ND filter to attenuate light, and a 250 us exposure. Here the centerof mass or spot separation stability is 396.02 nm for Noise Floor 1 and146.0 nm for Noise Floor 2. As the table illustrates, the stabilityimproves if 2D or 1D Gaussian filtering is added. Gaussian filters canbe added to mitigate the effect of fringes and provide a more uniformspot profile. As this table also illustrates, reducing the power of thelaser diode to 0.5 mW reduces the center of mass error, which meansgreater stability. Particularly, in this example, the center of masserror is reduced to 14.6 nm for Noise Floor 1 and 15.2 nm for NoiseFloor 2.

TABLE 1 Laser Power = 12 mW, Exp: 250 μs; ND filter 1D Gaussian 2DGaussian Filtering Laser Power = 0.5 mW, Filtering + (FWHM = 50 pix) +Exp: 250 μs Center of Mass Center of Mass Center of Mass Center of Mass(error in nm) (error in nm) (error in nm) (error in nm) Noise Floor 1 atx = 28, 396.02 113.05 64.76 14.6 y = −50 Noise Floor 2 at x = 28, 146.00134.66 46.30 15.2 y = 50

Operating the laser diode at 0.5 mW as opposed to 12 mW in this examplemeans that the laser is not truly in a lasing mode. This power level,however, is high enough that the laser diode is not operating in an ASEmode either. Instead, in this power range, the laser may be referred toas operating in a hybrid mode or a quasi-lasing mode. This is unusualfor laser operations. Normally, it is intended to run the laser in aclearly identifiable lasing mode, and pre-existing systems operate laserdiodes and power levels comfortably above the lasing threshold.Operating the laser in this hybrid mode is counterintuitive and atypicalof laser operations.

FIG. 31 is a diagram illustrating an example of the relationship of 5%full spectral width (FW at 5%) to laser power of various laser sources.As seen in this chart, the FW at 5% increases as the set powerdecreases. Accordingly, various examples are configured with the laserpower set to operate the laser in this hybrid mode to provide sufficientspot intensity for detection at the image sensor in a reasonable amountof time, yet to sufficiently limit the laser power so as to not createfringe patterns that introduce unwanted instability in the spotplacement. Because lower intensity requires a longer exposure time forsufficient readout at the image sensor, decreasing the laser power cannegatively impact the latency of the focus tracking system. Therefore,when determining whether sufficient intensity is provided, it may beuseful to consider the amount of time required to complete the focustracking measurement and whether that sufficiently achieves latencygoals for the system. The amount of power applied to the laser toachieve the foregoing depends on the laser diode specified, sensitivityand speed of the image sensor (for latency considerations), latencyrequirements of the system and the accuracy requirements of the system.

Other examples can be implemented with the laser power set to operatethe laser such that the dominant peak in the laser diode output at agiven frequency has a normalized intensity of between 15%-100% greaterthan any secondary peaks in the laser diode output. In yet otherexamples the power level at which the laser diode light source isoperated is selected such that the dominant peak in the laser diodeoutput at a given frequency has a normalized intensity of between15%-25% greater than a normalized intensity of a secondary peak in thelaser diode output. In still other examples the power level at which thelaser diode light source is operated is selected such that the dominantpeak in the laser diode output at a given frequency has a normalizedintensity of between 15%-100% greater than a normalized intensity of asecondary peaks in the laser diode output. In further examples, thepower level at which the laser diode light source is operated isselected such that the dominant peak in the laser diode output at agiven frequency has a normalized intensity of between 15%-200% greaterthan a normalized intensity of a secondary peak in the laser diodeoutput.

Another metric that can be used for setting the power at which the lightsource is operated can be the maximum exposure time the system cantolerate while meeting predetermined focus tracking latencyrequirements. Generally speaking, as the power at which the laser isoperated is reduced the amount of spot fringing is also reduced,improving focus tracking accuracy. However, below a certain power amountinsufficient intensity is provided at the image sensor to enabledetection of the spots or to enable detection in a sufficiently shortexposure time to meet latency requirements. Therefore, the power settingcan be reduced to the point where the required corresponding exposuretime is at or near the maximum exposure time allowed for system latencyin the focus tracking operation. In the example provided above, theexposure time for the light source operated at a 0.5 mW is 250 μs.

While various examples of the disclosed technology have been describedabove, it should be understood that they have been presented by way ofexample only, and not of limitation. Likewise, the various diagrams maydepict an example architectural or other configuration for the disclosedtechnology, which is done to aid in understanding the features andfunctionality that can be included in the disclosed technology. Thedisclosed technology is not restricted to the illustrated examplearchitectures or configurations, but the desired features can beimplemented using a variety of alternative architectures andconfigurations. Indeed, it will be apparent to one of skill in the arthow alternative functional, logical or physical partitioning andconfigurations can be implemented to implement the desired features ofthe technology disclosed herein. Also, a multitude of differentconstituent module names other than those depicted herein can be appliedto the various partitions. Additionally, with regard to flow diagrams,operational descriptions and method claims, the order in which the stepsare presented herein shall not mandate that the disclosed technology beimplemented to perform the recited functionality in the same orderunless the context dictates otherwise.

Although the disclosed technology is described above in terms of variousexample configurations and implementations, it should be understood thatthe various features, aspects and functionality described in one or moreof the individual examples are not limited in their applicability to theparticular example with which they are described, but instead can beapplied, alone or in various combinations, to one or more of the otherexamples of the disclosed technology, whether or not such examples aredescribed and whether or not such features are presented as being a partof a described example. Thus, the breadth and scope of the technologydisclosed herein should not be limited by any of the above-describedexamples.

Terms and phrases used in this document, and variations thereof, unlessotherwise expressly stated, should be construed as open ended as opposedto limiting. As examples of the foregoing: the term “including” shouldbe read as meaning “including, without limitation” or the like; the term“example” is used to provide example instances of the item indiscussion, not an exhaustive or limiting list thereof; the terms “a” or“an” should be read as meaning “at least one,” “one or more” or thelike; and adjectives such as “pre-existing,” “traditional,” “normal,”“standard,” “known” and terms of similar meaning should not be construedas limiting the item described to a given time period or to an itemavailable as of a given time, but instead should be read to encompasspre-existing, traditional, normal, or standard technologies that may beavailable or known now or at any time in the future. The term comprisingis intended herein to be open-ended, including not only the recitedelements, but any additional elements as well. Likewise, where thisdocument refers to technologies that would be apparent or known to oneof ordinary skill in the art, such technologies encompass those apparentor known to the skilled artisan now or at any time in the future.

The term “coupled” refers to direct or indirect joining, connecting,fastening, contacting or linking, and may refer to various forms ofcoupling such as physical, optical, electrical, fluidic, mechanical,chemical, magnetic, electromagnetic, optical, communicative or othercoupling, or a combination of the foregoing. Where one form of couplingis specified, this does not imply that other forms of coupling areexcluded. For example, one component physically coupled to anothercomponent may reference physical attachment of or contact between thetwo components (directly or indirectly), but does not exclude otherforms of coupling between the components such as, for example, acommunications link (e.g., an RF or optical link) also communicativelycoupling the two components. Likewise, the various terms themselves arenot intended to be mutually exclusive. For example, a fluidic coupling,magnetic coupling or a mechanical coupling, among others, may be a formof physical coupling.

The presence of broadening words and phrases such as “one or more,” “atleast,” “but not limited to” or other like phrases in some instancesshall not be read to mean that the narrower case is intended or requiredin instances where such broadening phrases may be absent. The use of theterm “component” does not imply that the elements or functionalitydescribed or claimed as part of the component are all configured in acommon package. Indeed, any or all of the various elements of acomponent, including structural elements, can be combined in a singlepackage or separately maintained and can further be distributed inmultiple groupings or packages.

It should be appreciated that all combinations of the foregoing concepts(provided such concepts are not mutually inconsistent) are contemplatedas being part of the inventive subject matter disclosed herein. Inparticular, all combinations of claimed subject matter appearing at theend of this disclosure are contemplated as being part of the inventivesubject matter disclosed herein.

The terms “substantially” and “about” used throughout this disclosure,including the claims, are used to describe and account for smallfluctuations, such as due to variations in processing. For example, theycan refer to less than or equal to ±5%, such as less than or equal to±2%, such as less than or equal to ±1%, such as less than or equal to±0.5%, such as less than or equal to ±0.2%, such as less than or equalto ±0.1%, such as less than or equal to ±0.05%.

Additionally, the various examples set forth herein are described interms of example diagrams and other illustrations. As will becomeapparent to one of ordinary skill in the art after reading thisdocument, the illustrated examples and their various alternatives can beimplemented without confinement to the illustrated examples. Forexample, block diagrams and their accompanying description should not beconstrued as mandating a particular architecture or configuration.

We claim:
 1. An imaging system comprising: a light source; a beamsplitter to create first and second beams from the light source; afocusing lens positioned to receive the first and second beams output bythe beam splitter, to focus the received first and second beams to aspot sized dimensioned to fall within a sample container to be imaged,and to receive first and second beams reflected from the samplecontainer; an image sensor positioned to receive the light beamsreflected from the sample container; and a roof prism positioned in theoptical path between the focusing lens and the image sensor, the roofprism dimensioned to cause the first and second beams reflected from thesample container to converge on to the image sensor.
 2. The imagingsystem of claim 1, further comprising a lens positioned to form a beamwaist along the optical path.
 3. The imaging system of claim 2, whereinthe light source comprises a laser and an optical fiber having first andsecond ends, the first end of the optical fiber to receive output fromthe laser, and wherein the second end is coupled to an insert that isslidable.
 4. The imaging system of claim 3, wherein moving the insertbetween a first position and a second position moves a beam waist alongthe optical path.
 5. The imaging system of claim 4, further comprising alocking mechanism to fix a position of the insert.
 6. The imaging systemof claim 1, wherein the beam splitter is a prism.
 7. The imaging systemof claim 1, further comprising a second light source, wherein the beamsplitter creates third and fourth beams from the second light source,and wherein the focusing lens is positioned (i) to receive the third andfourth beams output by the beam splitter, (ii) to focus the receivedthird and fourth beams to a second spot at the sample container, and(iii) to receive the third and fourth beams reflected from the samplecontainer.
 8. The imaging system of claim 7, further comprising a doveprism to cause the third and fourth beams reflected from the samplecontainer to converge with the first and second beams reflected from thesample container on to the image sensor.
 9. The imaging system of claim7, wherein the third and fourth beams are a look-ahead or look-behindfocus tracking set of beams.
 10. The imaging system of claim 1, whereinthe focusing lens is a prism.
 11. The imaging system of claim 1, whereinthe sample container comprises a plurality of layers.
 12. The imagingsystem of claim 11, wherein a top layer of the plurality of layers istransparent.
 13. The imaging system of claim 11, wherein the first andsecond beams are reflected from a surface of the plurality of layers ofthe sample container.
 14. The imaging system of claim 11, furthercomprising a blocking structure positioned to block reflections of thefirst and second beams from at least one surface of the plurality oflayers.
 15. The imaging system of claim 1, wherein the focusing lens ismoveable from a first position to a second position, wherein thefocusing lens is to focus the received first and second beams at a firstsurface within the sample container when in the first position, andwherein the focusing lens is to focus the received first and secondbeams at a second surface within the sample container when in the secondposition.